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Use of capillary electrophoresis to determine a selected group of flavonoids
Tran Xuan, Tiep ; Mravcová, Ludmila (referee) ; Řezáčová, Veronika (advisor)
In recent years, the use of natural medicinal properties of plants has again come to the forefront of interest of the public. Because of their therapeutic properties, these plants are commonly used in alternative treatments for minor ailments. This thesis deals with the determination of polyphenolic acids in extracts from aronia, echinacea, tetradenia and centella. One of the aims of the thesis was to compare the amount of polyphenolic acids in individual plants with their antioxidant properties. The theoretical part includes a general introduction to flavonoids, introduction to polyphenolic acids, introduction to the principles of capillary zone electrophoresis and a summary of selected foreign studies for the determination of flavonoids. An important chapter of the theoretical part is a description of individual plants with an explanation of their possible therapeutic use. The experimental part is devoted to the process of optimization of electrophoretic determination of selected polyphenolic acids and evaluation of their content. It was found that in the extracts six representatives of polyphenolic acids occur in minimal amounts, often below the limit of quantification or detection of the method.
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Multidimensional Liquid Phase Separations
Šesták, Jozef ; Čáslavský, Josef (referee) ; Pulkrabová, Jana (referee) ; Česla, Petr (referee) ; Kahle, Vladislav (advisor)
This dissertation is dedicated to the topic of multidimensional liquid phase separations. This separation techniques are developed for analysis of complex samples containing thermally labile, low volatile or high molecular weight components that can´t be analysed by two-dimensional (2D) gas chromatography. Concepts of peak capacity and orthogonality are explained and various methods of their determination are stated in theoretical part of dissertation. High performance column liquid chromatography (HPLC) and high performance capillary electrophoresis (HPCE) are suggested as the most suitable methods for automated multidimensional liquid phase separations on-line coupled to mass spectrometry. Configuration of simplified miniaturized liquid chromatograph is described in experimental part of this thesis. Original concept of the system has been extended by simple mobile phase gradient generation technique. Correct function was demonstrated on repeatable separation of alkylphenones, peptides, nitroaromatics, and nitroesters. This system has been utilized as a base for a couple of simple two-dimensional separation platforms for HILIC-MALDI-MS analysis of glycans, for separation of peptides based on off-line coupling of isoelectric focusing and capillary liquid chromatography, and finally for on-line IEC×RPLC, RPLC×RPLC, and HILIC×RPLC two-dimensional liquid chromatography. Correct operation of submitted platforms has been proved.
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Vliv zpracování kávy na obsah kofeinu a jeho účinky na lidský organismus
Odehnalová, Barbora
This diploma thesis focuses on the comparison of caffeine content in coffee processed in different ways. It involves dry and wet processing, where in the first mentioned case the coffee beans are dried in the sun to the ideal moisture level after collection and sorting. In the wet method, the coffee is fermented in vats with added microorganisms and then dried. The work also includes the effects of caffeine on the human organism.
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Detekce nukleových bazí pomocí kapilární elektroforézy
Hendrychová, Kateřina
Capillary electrophoresis (CE) is one of the most important techniques used for high-throughput separations in modern analytical chemistry. Its main advantages include the possibility of using different detection techniques, speed and efficiency of separation as well as reproducibility. These properties are used in the separation of a wide variety of analytes ranging from inorganic ions to nanoparticles and were also verified in this work, in which the basic building blocks of DNA and RNA, nucleic bases, which serve as biomarkers for many disorders, diseases and food safety, were measured using capillary electrophoresis with a UV detector. The bachelor thesis is focused on the development and optimization of a method to detect and separate 5 nucleobases and purine. The separation was achieved by using 40 mM borate buffer pH 9,8 with the addition of 60 mM SDS as a detergent which forms micelles.
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Fluorescenční sondy pro detekci sacharidů a glykovaných proteinů pomocí kapilární elektroforézy
Kozáková, Silvie
Glycated albumin (gHSA) and glycated hemoglobin (HbA1c) represent important biomarkers assessing the progression of diabetes mellitus disease. The aim of this thesis was to develop and optimize a method for the simultaneous determination of these glycated proteins from human blood by capillary electrophoresis using the fluorescent probe ABAM (anthracene boronic acid methacrylate). Thanks to this new method, it was possible to selectively determine these two proteins with a resolution of 5.70 and the LOD of 11.9 µg/ml for gHSA and 26.5 µg/ml for HbA1c. Subsequently, the optimized method was successfully applied to a real human blood sample. The simultaneous determination of these biomarkers can help to better monitor dynamic changes during diabetes and enable its monitoring in cases, where HbA1c assessment is inappropriate for medical reasons (anemia, etc.).
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Development of a new DNA mutation detection technology for application in cancer diagnostics
Janoušková, Kristýna ; Minárik, Marek (advisor) ; Klepárník, Karel (referee)
The aim of this diploma thesis was to test a new technology for DNA mutation detection and to optimize the parameters related to the introduction of a new method, that uses this technology, into oncological diagnostic practice. The new method consists of an experimental modification of an already used method for the detection of somatic mutations in tumor DNA, which is based on heteroduplex analysis and denaturing capillary electrophoresis. The new aspect of the technology is the use of short oligonucleotide probe, which significantly simplifies the interpretation of data and also enables the simultaneous examination of multiple mutations in one reaction (multiplexes). Key words: DNA, denaturating capillary electrophoresis, heteroduplex, mutation, PCR, probe
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On-line derivatization of saccharides in capillary electrophoresis
Mareš, Vít ; Křížek, Tomáš (advisor) ; Coufal, Pavel (referee)
Saccharides chains may suffer from various inaccuracies during their biosy- thesis, that effects their biologic functions. Therefore, they can be used for dia- gnosis of some diseases or quality control in biopharmaceutics. Capillary electro- phoresis has its place in diagnosing these occuring errors. In this thesis a method of on-line derivatization of saccharides in fused-silica capillary with 50 cm in lenght and 50 µm in inner diameter was developed. Gluco- se was used as a model molecule, which was labeled by a fluorescent derivatization reagent, 7-aminonaphthalene-1,3-disulfonic acid. Derivatization of glucose and its separation needed optimalization. First was determined an electrophoretic mo- bility of the derivatization reagent in 0.5; 1.0 and 2.5M acetic acid and 20mM phosphate buffer (pH = 3.5). Both, 2.5M acetic acid and 20mM phosphate buffer, were tested as solvents for 7-aminonaphthalene-1,3-disulfonic acid. The reaction with glucose was the best in acetic acid solution. On the contrary, phosphate buf- fer was found to be the best background electrolyte for the separation. Derivatized glucose was detected by UV detector at 229 nm. Henceforth, the reaction con- ditions were adjusted such as reaction time, voltage and time of its application. Input voltage was applied negative due to the...
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Determination of glutathion in plant samples using capillary electrophoresis
Skála, Michael ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
This work is based on a previously published method for determination of both glutathione forms by capillary zone electrophoresis equipped with UV spectrometric detector. Theoretical part described biological and biochemical functions of glutathione as a molecule. It mentioned historically used methods for glutathione determination, their accuracy, precision and reliability and focused on electrophoretic methods, especially capillary zone electrophoresis. Basic principles of capillary zone electrophoresis were described, followed by an overview of approaches to treatment of different types of samples, with special focus on plant samples. At the end, method this work was based on was described. The experimental part describes the development and optimization of this method. Firstly, optimum chemical composition and ionic strength of background electrolyte were determined - two electrolytes were compared, with NaCl and without NaCl - no significant differences were observed, so NaCl was excluded from BGE. Next, optimum concentration of hexadimethrine bromide was determined. The effect of HDB concentration on electroosmotic flow was investigated. Calibration curve for different concentrations of analytes in water was constructed and repeatability, LOD and LOQ of the method were determined....
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Modification of inner capillary surface for electrophoretic separations of liposomes
Píplová, Renata ; Křížek, Tomáš (advisor) ; Čokrtová, Kateřina (referee)
Electroosmotic flow is one of the main factors that affect the resolution of separations in capillary electrophoresis and its modification is often necessary. One way of modification of the electroosmotic flow is the use of dynamic or permanent coating of the inner capillary surface. Such modifications during liposome separation can also reduce the tendency of liposomes to adsorb to the capillary surface. The aim of this work was to find a stable coating that will sufficiently eliminate the electroosmotic flow and to test its effect on the separation of liposomes. At first, the dynamic coatings of the capillary with Pluronic F-127, polyvinylpyrrolidone K30, polydiallyldimethylammonium chloride, polyethylene glycol and polyvinyl alcohol were tested using four different methods. Individual polymers and methods differed in the rate of elimination of the electroosmotic flow as well as in the stability of the capillary coatings. Dynamic coatings with Pluronic F-127, polyvinylpyrrolidone K30 and polydiallyldimethylammonium chloride were selected for further liposome separation testing. Fluorescently labeled liposomes were separated using capillary electrophoresis with laser-induced fluorescence detection and the mobilities of liposomes separated using capillaries with different coatings were determined....
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Comparison of detection techniques for analysis of saccharides by capillary electrophoresis
Vlčková, Nikol ; Křížek, Tomáš (advisor) ; Kozlík, Petr (referee)
The aim of this work was to compare detection techniques of capillary electrophoresis to determine saccharides. The techniques were contactless conductivity detection, direct and indirect UV detection and fluorescence detection. Determined saccharides were glucose, fructose and sucrose, lactose was employed as internal standard. For conductivity detection the capillary has internal diameter of 20 µm. Voltage applied to capillary was 15 kV and temperature was 25 řC. 40mM NaOH was used as BGE. The calibration dependence was measured between 0,005 - 0,5 mg/ml. Repeteabilityof peak areas and migration times was measured in two concentration levels of 0,02 and 0,2 mg/ml. Detection and quantification limits were between 1,0 - 5,8 µg/ml. RSD values for repeatability of peak areas and migration times were between 0,01- 8,64 %. Difference in recovery from real concentration of analytes was between 7 - 22 %. For other detection methods the capillary had inner diameter of 50 µm. Voltage applied to capillary for indirect UV detection was 28 kV and temperature was 30 řC. Wavelength for detection was 207 nm and 50mM glycylgylycine with pH value of 12,8 was used as BGE. The calibration dependence was measured between 0,1-1 mg/ml. Repeteability of peak areas and migration times was measured in two concentration...
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